The present invention concerns cloning vectors for heterologous genes in yeasts as well as the yeast strains transformed by said vectors.
In more detail, the present invention concerns vectors for the transformation of yeasts, in which vectors the entire nucleotide sequence of the pKD1 plasmid of Kluyveromyces drosophilarum or a part of the same is present. Moreover, the present invention concerns specifically yeasts providing functions for a replication of these vectors.
As is well known, the transformation of yeasts up to the present time has been limited to certain species because of the poor availability of the yeast plasmids that are required for the construction of transformation vectors (Gunge, N., Ann. Rev. Microbiol. 37, 253-276 (1983); Toh-e, A., Tada, S. and Oshima, Y., J. Bacteriol. 151, 1380-1390 (1982): Toh-e, A., Arakl, H., Utatsu, I., and Oshima, Y., J. Gen. Microbiol. 130, 2527-2534 (1984).
The 2.mu. plasmid isolated from Saccharomyces cerevisiae has been, up to the present time, the only plasmid successfully employed for the transformation of said S. cerevisiae. However, vectors derived from the 2.mu. plasmid have shown a low transformation efficiency and/or a limited ability to replicate stably in yeasts other than Saccharomyces cerevisiae.
Accordingly, it was quite evident that plasmids for the construction of vectors suitable for the transformation of other yeasts were needed, especially for yeasts of industrial importance, including, for example, those belonging to the genus Kluyveromyces.
However, all attempts to date to satisfy this need have not been very effective.
More specifically, two vectors have been used for the transformation of Kluyveromyces lactis: one contained a chromosomal DNA segment of K. lactis (Das, S. and Hollenberg, C. P., Current Genetics 6 123-128 (1982), and the other was derived from the linear plasmid pGK1 also isolated from K. lactis (L. de Louvencourt, H. Fukuhara, H. Heslot, M. Wesolowski, French Patent Application No. 8209564 of ELF Biorecherche; L. de Louvencourt et al., J. Bacteriol. 154, 737742 (1982).
It is important to point out that, although both of the vectors mentioned above contain nucleotide sequences of K. lactis that allow their replication in this yeast, the efficiency and/or the stability with which transformed clones are obtained is quite low.
Accordingly, an object of the present invention is the production of cloning vectors for yeasts, in particular for the genus Kluyveromyces, which have improved transformation efficiency and which show a higher stability in the transformed cells.
Now it has been surprisingly found, according to the present invention, that this object can be attained by using as an essential part of the transformation vectors, a plasmid or at least a genetic element thereof discovered in a strain of the yeast Kluyveromyces drosophilarum, in the course of a research project on new plasmids (C. Falcone, et al Plasmid, 15 248-252(1986)).
The plasmid dealt with herein consists of a circular duplex DNA molecule of 1.6 micron circumference, which was found in the UCD 51-130 Kluyveromyces drosophilarum strain (U.C.D. Collection, University of California, Davis, Calif. 95616). The same strain is available from the American Type Culture Collection (U.S.A.) under the accession No. ATCC 56496, from the Agricultural Research Service Culture Collection (U.S.A.) under the No. NRRL Y-8278 and from the Centraalbureau voor Schimmelcultures (The Netherlands) under the No. CBS 2105.
This plasmid, which is called pKD1, is present in the cells in a high copy number (70-100 per cell) and it is easily separable from the chromosomal DNA by employing standard procedures.
The complete nucleotide sequence of pKD1 has been determined, and so the exact size of the plasmid (4757 base pairs) is known. In addition, the analysis of the sequence confirmed the data previously obtained through DNA-DNA hybridization concerning the substantial difference in primary structure between this plasmid and the 2.mu. and pGK1 plasmid.
The plasmid exists inside the same cell in two interchangeable molecular forms called A and B, which are present in equal amounts and differ from one another by the orientation of a central segment of 2.15 kilobases that, in FIG. 1 enclosed herein, is comprised between two vertical dashed lines. FIG. 1 shows the localization of the sites for numerous restriction enzymes (such location referring to the A and B forms of pKD1 when drawn as linear molecules), such enzymes being indicated in the left margin of FIG. 1. The scale shown in the horizontal direction points out the distance in kilobases (kb) between these sites on the plasmid. Numbers shown inside the segments indicate their respective lengths in base pairs. The asterisks point out those fragments whose localization was uncertain before the entire sequence was determined.
The analysis of the sequence showed the presence in this plasmid of a DNA segment of 346 base pairs which was found again, identical in sequence but with inverted orientation, at a distance of 2136 nucleotides.
This inverted and repeated (IR) sequence is likely to be fundamental in the mechanism of interconversion between the A and B forms of pKD1, in analogy with what has been observed with the 2.mu. plasmid of S. cerevisaiae.
From the nucleotide sequence of the plasmid pKD1 the presence of three possible genes, called A, B and C, was deduced (with respective lengths of 1341, 1245 and 636 nucleotides); these genes, based on sequence homologies and preliminary studies on their functions, seem to be analogous, respectively, to the FLP, Rep 1 and Rep 2.mu. genes that are present in the 2 plasmid (Broach, J. R., Cell 28, 203-204 (1982)).
The pKD1 plasmid can be advantageously employed for the construction of cloning vectors in yeasts, primarily because its circular form allows simple manipulations. Moreover, this plasmid contains numerous unique sites for restriction enzymes, such as EcoRI, ClaI and PstI, and this feature is also particularly advantageous for the insertion of heterologous genes and for the construction of recombinant vectors.
The presence of the pKD1 plasmid and of the vectors derived from the same in the yeast cells at a high copy number is a further advantage because it allows an amplification of the inserted heterologous gene.
Moreover, it is advantageous that neither the integrity nor the continuity of the sequence of the pKD1 plasmid is needed for the construction of stable vectors at a high copy number according to the present invention. Indeed, it is possible to insert the gene to be cloned specifically into one of the unique restriction sites, so interrupting the pKD1 sequence, or to use just a part of this plasmid, containing the replication origin and a segment containing a cis-acting stabilizing sequence (or locus), called CSL.
The specific object of the present invention consists in the construction of cloning vectors for heterologous genes in yeasts, such vectors comprising the entire DNA of the pKD1 plasmid (isolated from Kluyveromyces drosophilarum) or a part thereof including at least one genetic element and a DNA segment bearing a heterologous gene and including the sequences that insure the expression of said gene such sequence being derived also from pKD1.
When the entire DNA of the pKD1 plasmid is employed, it is preferable that the gene to be cloned be inserted into one of the unique restriction sites, in particular into the EcoRI or the PstI site.
Among the genes that are most suitable for cloning, the URA3 gene of S. cerevisiae is in particular worth considering, preferably as a part of the YIp5 plasmid, which consists of the sequence of the pBR322 plasmid of Escherichia coli and, in addition, of the sequence of this URA3 gene.
In another preferred form of the present invention (vector A15), the vector contains the 1717 bp segment of DNA of the B form of the pKD1 plasmid obtained with the enzyme BamHI, inserted into the unique BamHI site of the YIp5 plasmid.
In another preferred embodiment of the present invention (vector HP1), the vector contains the 375 bp DNA segment of the pKD1 plasmid obtained by digestion with the HinPI enzyme, inserted into the unique ClaI site of the YIp5 plasmid.
In a further preferred embodiment (vector HCSl), the cloning vector contains the 207 bp segment of the pKD1 DNA obtained with the restriction enzymes BclI and PvuII, replacing the BamHI-NruI segment of the tetracycline resistance gene of the HP1 vector.
The present invention also relates to yeasts transformed by the above-mentioned vectors and, particularly but not exclusively, to the strains of the genus Kluyveromyces and more specifically of the species Kluyveromyces lactis. While the Kluyveromyces genus is of particular interest since it has been found that the pKD1 plasmid can replicate in the Kluyveromyces strains with variable stability, the present invention is intended to cover the transformation of all yeasts in which the pKD1 plasmid is able to replicate, whether or not such yeast is classified at a given time under the Kluyveromyces genus. For example, it has been demonstrated that yeasts which have been classified as Candida are actually Kluyveromyces. Martini A. V. and Martini A., Int. J. Syst. Bacteriol. Vol. 37, p. 380-385, 1986, reported DNA-DNA reassociation experiments which demonstrated that C. macedoniensis and K. Marxianus have high DNA sequence homology and Sor F. and Fukuhara H., Yeast, Vol. 5, p. 1-10, 1989, confirmed that hypothesis by analysis of chromosomal migration patterns and mitochrondrial DNA.
It is interesting to note that yeasts transformed according to the present invention can be advantageously exploited for the production of the protein coded by the heterologous gene inserted into the vector. Such yeasts can be, for example, of remarkable importance in the foodstuff field (the production of amylase, rennin, pectinase and so on) or in the pharmaceutical field (insulin, interferon and so on).
In addition, such transformed yeasts can constitute in themselves integrating products for the feeding of animals or livestock (biomass).
For the sake of discussion, and for illustrative but not limitative purposes, the use of the invention for the cloning of the URA3 gene of Saccharomyces cerevisiae in a uraA strain of Kluyveromyces lactis will be considered. The uraA strain is normally unable to grow in uracil-free medium. The URA3 gene that is present in vectors derived from the pKD1 plasmid was shown to be expressed in the host cell and such cells were shown to be able to grow in uracil-free mediums. This shows that the orotidine-monophosphate decarboxylase protein encoded by the URA3 gene of S. cerevisiae is effectively synthesized by K. lactis.